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Cancer Research Journal 2016; 4(4): 58-68
http://www.sciencepublishinggroup.com/j/crj
doi: 10.11648/j.crj.20160404.11
ISSN: 2330-8192 (Print); ISSN: 2330-8214 (Online)
Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
Nandini N. Manoli1, Nandish S. Manoli
2, Lopamudra Kakoti
1, Akshata Kamath
1, Shweta Kulkarni
1
1Dept of Pathology, Jagadguru Shri Shivarathreswara Medical College, Jagadguru Shri Shivarathreswara University, Mysore Karnataka,
India 2Dept of OBG, JSS Medical College, JSS University, Mysore Karnataka, India
Email address: [email protected] (N. N. Manoli), [email protected] (N. N. Manoli)
To cite this article: Nandini N. Manoli, Nandish S. Manoli, Lopamudra Kakoti, Akshata Kamath, Shweta Kulkarni. Manual Liquid Based Cytology in Diagnosis
of Gynecologic Pathology. Cancer Research Journal. Vol. 4, No. 4, 2016, pp. 58-68. doi: 10.11648/j.crj.20160404.11
Received: February 18, 2016; Accepted: March 21, 2016; Published: June 13, 2016
Abstract: Abnormal uterine bleeding (AUB) is a commonly encountered complaint in gynecology department. Endometrial
cancer is the fourth most common malignancy in women and the most frequent gynecological cancer in developed countries.
With 5,28, 000 new cases every year, cervical cancer is the fourth most common cancer affecting women worldwide, after
breast, colorectal, and lung cancers. Though the cytological examination has been the mainstay for early detection of cervical
cancer, and found to be useful in detection of endometrial cancer, its widespread use has not been possible in the developing
countries due to paucity of resources, man power and other facilities Its sensitivity reduces to less than 50% when there is
presence of obscuring blood, inflammation or thick areas of overlapping epithelial cells. Manual Liquid Based Cytology
(MLBC) is a cost effective technique that enables cells to be suspended in a monolayer and thus improve detection of precursor
lesions and specimen adequacy. The residual sample can be used for other tests like Cell block and immunocytochemistry.
Objectives: 1. To improve the diagnosis of gynecological cancers by a method called as Manual Liquid Based Cytology
(MLBC). 2. To study a cost effective method of studying both endometrial and cervical cancer with help of ancillary
techniques like cellblock, immunocytochemistry 3To compare the findings between conventional pap smear (CPS) and MLBC
in detection of gynecological conditions of endometrium and cervix. Methodology: Samples were collected using Ayres
spatula by split sample technique from transformation zone of cervix which included outpatients of gynecology dept. The
women were aged between 20-60 years, 82 cases with bleeding history were taken to study endometrial pathology, while 100
cases of white discharge per vagina were selected to study the cervix. 60 cases were later subjected to ancillary studies like
cellblock with Immunocytochemistry. Histopathological correlation was done for cases wherever possible. Results: The study
on endometruim showed MLBC is more sensitive and specific than CPS in diagnosis of malignant lesions. The contingency
coefficient for LBC/Histopathology V/s CPS/Histopathology was 0.572 V/s 0.556. It was observed for cervical lesion that
increased detection rate was 150% for low grade intraepithelial lesion. The Positive predictive value for diagnosing neoplastic
lesions on cell blocks was 75%, while Concordance Rate of CB/Histopathology Vs CPS/Histopathology was 74% vs. 54%.
Conclusion: MLBC is a cost effective method for detections of cancerous lesions of endometruim and cervix.
Keywords: Manual Liquid Based Cytology, Endometrium, Cervix
1. Introduction
Endometrial carcinoma (EC) is the commonest malignancy
of the female genital tract in the USA and Europe, accounting
for nearly 50% of all new diagnoses of gynecological cancer
[1, 2, 3]. The incidence of EC increased from 13.5 to 20.3 per
100, 000 between 1993 and 2010 in the UK, [4] and the
incidence and mortality have been reported to have increased
by 1.1% and 0.3%, respectively, annually during the same
period of time in the USA [5]. The incidence of EC is also
increasing in Japan, even in younger age groups, [6] where it
has become more frequent each year over a period of 30
years, and as a proportion of all uterine carcinoma rose from
10% in 1983 to 45% in 2004 [7]. The main risk factors for
EC are obesity, diabetes, estrogen use, tamoxifen treatment
59 Nandini N. Manoli et al.: Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
polycystic ovarian syndrome (PCOS), a history of infertility,
alcohol abuse and antidepressant agents [8, 9, 10, 11]. Many
of these factors are tightly linked to current lifestyles in
developed countries. A small but significant percentage
(about 2-3%) of EC is attributable to Lynch syndrome, a
hereditary cancer predisposition syndrome which
significantly increases the risk of colorectal, endometrial and
other cancers. [1, 2] Fig-1
With 528, 000 new cases every year, cervical cancer is the
fourth most common cancer affecting women worldwide,
after breast, colorectal, and lung cancers; it is most notable in
the lower-resource countries of sub-Saharan Africa. It is also
the fourth most common cause of cancer death (266 000
deaths in 2012) in women worldwide. [12] Almost 70% of
the global burden falls in areas with lower levels of
development, and more than one fifth of all new cases are
diagnosed in India. Fig-2
Fig. 1. Graph showing the incidence of endometrial carcinoma with age and ethnicity.
Fig. 2. Worldwide incidence of cervical cancer.
The main focus is on the secondary prevention of cervical
cancer through early detection, a focus point of National
Cancer Control Programme revised in 1985 [13]. Though the
cytological examination has been the mainstay for early
detection of cervical cancer, its widespread use has not been
possible in the developing countries due to paucity of
resources, man power and other facilities. Moreover,
although Conventional Pap Smears (CPS) screening leads to
reduction in the rate of invasive cancer of the uterine cervix,
its sensitivity reduces to less than 50% when there is
presence of obscuring blood, inflammation or thick areas of
overlapping epithelial cells [14, 15]. These problems with the
CPS, gave rise to the advanced technologies, like, Thin Prep
and Sure Path commonly used in the setup of developed
countries, like UK and USA [15, 16]. Use of these
technologies however are quite resource intensive and
therefore not feasible in the setup of developing countries.
On the other hand, Manual Liquid Based Cytology
(MLBC) is a technique that enables cells to be suspended in a
monolayer and thus improves detection of precursor lesions
and improvement of specimen adequacy. MLBC has been
reported to improve the effectiveness of cervical cancer
screening in a population by increasing the detection of
histologically confirmed neoplastic and preneoplastic disease
while simultaneously decreasing over diagnosis of benign
processes [17]. Also, in case of MLBC, the residual sample
can be used for other tests like detection of HPV, DNA and
immunocytochemistry thereby increasing the utility of
MLBC [14, 15].
Cancer Research Journal 2016; 4(4): 58-68 60
There are studies [15, 18] which have dealt with liquid
based cytology and have found its diagnostic accuracy
comparable with conventional Pap smears. MLBC method
however is specific to the laboratory, available equipments,
fixatives and polymer solutions, etc. Therefore the overall
aim of our study was to assess the utility of indigenous
MLBC in comparison of CPS for low resource settings.
Specific objectives of the study were: a) To compare the
morphological view of different diagnoses according to CPS
and MLBC, b) To compare the cellular and nuclear
parameters according to two methods and c) To compare the
validity of the two methods in terms of sensitivity and
specificity.
As MLBC can be used for ancillary studies like cell block
study, immunocytochemistry and HPV testing, one of the
ancillary techniques which we studied is cell blocks prepared
from residual tissue fluids and fine-needle aspirations which
can be useful adjuncts to smears for establishing a more
definitive cytopathologic diagnosis. [19, 20].
Thus, our study includes MLBC in Endometrium &Cervix
with an ancillary technique, cell block to improve the
diagnostic accuracy of gynecological conditions in women.
2. Material & Methods
The present study was undertaken to observe the presence
of endometrial cells in cervical cytology smears using
conventional Pap smears and the manual method of liquid-
based cervical cytology (MLBC) in all abnormal uterine
bleeding cases.
Samples were collected using the split-smear technique
from 82 patients (sample size) in the age group of 20 to 60
years attending the gynecology out-patient clinic at JSS
Hospital, Mysore, prospectively from July 2012 to July 2014
for a period of two years.
All the patients were clinically examined in detail
according to the proforma and relevant radiological findings
were collected. Material collected was stained by Pap stain.
Ayre’s plastic spatula was used to scrape the cervix. It was
immediately put into a vial containing the fixative solution.
The fixative contained 0.5 gm of sodium chloride, 0.5 gm of
sodium citrate, 50 ml of 10% formalin and 50 ml of isopropyl
alcohol in 100ml. The sample was collected and mixed with
equal parts of fixative. It was centrifuged at 2000 rpm for 5
minutes. The supernatant was decanted and excess fixative
was blotted 1-2 ml of polymer solution (containing 2 gm of
agarose, 10 ml of polyethylene glycol, 2 ml of poly-L-lysine
and 88 ml of distilled water) was added to the deposit. It was
again centrifuged at 2000 rpm for 5 minutes. The deposit was
pipetted in a circular motion on to a glass slide. The slides
were placed on a metal tray and dried in an hot air oven at 50
degrees (Indirect heat fixation) and further fixed by dipping
in 95% alcohol for 5 minutes. Stained with the Conventional
Pap stain.
Simultaneously, scrape smears were also collected using
Ayre’s spatula. The scrapings were evenly spread onto a glass
slide, and immediately fixed in ethyl-alcohol fixative (95%
ethylalcohol). After fixation, smears were stained using
conventional Pap stain.
Both smears were screened and results were compared.
Cyto-histologic correlation was done. The Bethesda system
2001 was used for reporting cervical cytology for both
groups.
The cases in which a D&C or hysterectomy was done,
specimens were collected in 10% formalin and allowed to fix
overnight. Detailed gross examination was done and bits
were given. Paraffin embedded H and E stained sections were
obtained and studied under light microscopy.
Similarly another study on cervical pathology was
conducted Samples were collected using the split-smear
technique from 100 patients (sample size) in the age group of
20 to 80 years attending the gynaecology out-patient clinic at
JSS Hospital, Mysore. All the patients were clinically
examined in detail according to the proforma and details of
other relevant laboratory investigations were collected only if
necessary. Material collected was stained by Pap stain. The
cases collected were patients with clinical suspicion of
cervical pathology.
The processing of the samples was done as in the study on
Endometrial pathology. Both smears were screened and
results were compared. Cyto-histologic correlation was done
in those cases in which a colposcopic biopsy was also done.
The Bethesda system 2001 was used for reporting cervical
cytology for both groups.
Whenever possible, ancillary techniques were applied for
preparations of cell blocks and HPV- DNA testing from the
residual cytocentrifuged sample.
The cases in which a colposcopic biopsy or hysterectomy
was done, specimens were collected in 10% formalin and
allowed to fix overnight. Detailed gross examination was
done and bits were given. Paraffin embedded H and E stained
sections were obtained and studied under light microscopy.
To extend our work on ancillary techniques we took the
study further, to work on cell block as ancillary technique
with IHC on cell blocks whenever necessary. The cell blocks
studied were lesser than the liquid based cytology cases.
The study was undertaken to prepare the cell blocks from
samples of manual liquid based cytology (MLBC) and to
compare it with conventional pap smears and liquid based
smears and correlate with histopathology wherever possible.
Samples of MLBC of which cell block preparation was
made were collected from about 60 patients in the age group
of 20 to 70 years with gynecological complaints like white
discharge P/V and bleeding P/V attending the Gynecology
out-patient clinic at JSS Hospital, Mysore. In the present
study KI 67 and p16 markers were done on cell block
preparation.
3. Results
In the present study, 82 cervical smears prepared by
conventional pap and MLBC were studied, out of which 52
cases (63.4%) were reported with the presence of endometrial
cells, Among the 52 cases 38 cases were identified as benign
61 Nandini N. Manoli et al.: Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
endometrial cells and 14 cases as atypical/ malignant
endometrial cells.
3.1. Conventional Pap Smears vs Manual Liquid Based
Cytology
Among the 52 cases with endometrial cells, 46 were
detected in conventional Pap smear and 38 cases in Manual
Liquid Based Cytology. Out of 46 cases of endometrial cells-
35 were benign and 11 were atypical cells in conventional
pap smear, while in manual liquid based cytology 28 were
benign and 10 were atypical endometrial cells out of 38
cases. (Table 1)
Table 1. Distribution of cases into benign and Atypical/malignant
endometrial cells.
Lesions No. Percentage (%)
Benign endometrial cell 28 53.9
Atypical/malignant endometrial cells 10 19.2
Absent 11 21.2
Inadequate 03 05.7
Total 52 100
3.2. Morphological Distribution of Histopathological
Examination
On follow up with histopathology, maximum 8 cases of
leiomyoma with proliferative endometrium, followed by 6
cases of simple hyperplasia without atypia and 4 cases each
of adenomyomatous polyp and endometrial carcinoma type 1
were diagnosed. There was only one case each of
endometrial type 2 carcinoma and complex hyperplasia with
atypia and 3 cases each of squamous cell carcinoma,
endometritis and disordered proliferative endometrium.
Remaining cases were 2 proliferative endometrium and 1
case of placental site trophoblastic reaction. (Table 2) FIG 3
Table 2. Morphological distribution of histopathological examination of
endometrial pathology.
Histopathological diagnosis of
endometrium No. of cases Percentage (%)
Proliferative phase 02 5.1
Endometritis 03 7.7
Disordered proliferative phase 03 7.7
Leiomyoma 08 20.5
Simple hyperplasia without atypia 06 15.4
Complex hyperplasia with atypia 01 2.6
Adenomyomatous polyp 04 10.2
Atrophic endometrium 03 7.7
Endometrial carcinoma type 1 04 10.2
Endometrial carcinoma type 2 01 2.6
Squamous cell carcinoma 03 7.7
Placental site reaction 01 2.6
Total 39 100
a b
c d
Fig. 3. a. Proliferative growth occupying the entire uterine cavity b. CPS-Atypical Endometrial Cells - Smear shows cells with high N/C ratio arranged in
clusters with enlarged hyperchromatic nuclei and fairly abundant cytoplasm. (Pap, X400) c. MLBC Atypical Endometrial Cell - Smear shows cells with high
N/C ratio arranged in clusters with enlarged hyperchromatic nuclei and fairly abundant cytoplasm(Pap, X400) d. HPE - Endometrial Adenocarcinoma type 1
-Section shows a tumor displaying features of endometrial adenocarcinoma. (H & E, X10).
Cancer Research Journal 2016; 4(4): 58-68 62
3.3. Conventional Pap Smear Findings in Correlation with
Histopathology
� Histopathological study was obtained in 29 out of 35
cases with benign endometrial cells and 8 out of 11
cases with atypical/malignant endometrial cells.
� Among them, 21cases and 6 cases were consistent with
histopathological findings of benign and malignant
endometrial pathology respectively.
3.4. Manual Liquid Based Cytology Findings in
Histopathology Correlation
� Histopathological study was obtained in all the 29 cases
with benign endometrial cells and 8 out of 10 cases with
atypical/malignant endometrial cells.
� Among them, 20cases and 6 cases were consistent with
histopathological findings of benign and malignant
endometrial pathology respectively
� MLBC is more sensitive and specific than CPS in
diagnosis of malignant lesions.
� The contingency coefficient by LBC/Histopathology
V/s CPS/Histopathology was 0.572 V/s 0.556.
Results of study of CPS vs MLBC for cervical cancer
screening showed the following,
Panel of various cellular and nuclear parameters were
compared between CPS and MLBC (Table 3).
Table 3. Morphological Features as Observed Through Conventional Pap
Smear (CPS) and Manual Liquid Based Cytology (MLBC) Methods.
Morphological feature CPS MLBC
Cellularity Unsatisfactory in 10 cases Adequate
Cleanliness of background Absent Absent
Uniformity of distribution present Absent
Artifacts present Rare
Cellular overlapping Yes (Marked) Yes (Rare)
Architectural change Yes Rare
Cellular morphological Change yes No
Nuclear change Not always clear Always very
clear
Cellularity was adequate in all of the MLBC cases whereas
it was unsatisfactory in many CPS cases. The background
was observed to be clean in all cases of MLBC which was
not the case in majority of CPS. Uniform distribution seen in
MLBC with cellular overlapping was seen more in CPS than
in MLBC. Artifacts were present in most CPS samples.
Architectural and cellular morphologic changes were present
in most of CPS samples. Inflammatory infiltrate were
prominently present in CPS but decreased in MLBC cases.
Nuclear changes were very clear by MLBC, but not so clear
by CPS. Diagnostic features of 100 cases according to both
CPS and MLBC were divided into 10 categories. FIG 4&5
a b c
Fig. 4. a. CPS-Inflammatory smear (NILM) (PAP, X200) b. MLBC-Inflammatory smear with clear background (PAP, X200) c. HPE-section studied shows
chronic cervicitis. (H&E, X200).
a b c
Fig. 5. a. CPS-squamous cell carcinoma of cervix (PAP, X200) b. MLBC-Squamous cell carcinoma of cervix. with clear background. (PAP, X200). c. HPE-
squamous cell carcinoma of cervical biopsy. (PAP, X200).
63 Nandini N. Manoli et al.: Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
In the study, the comparison between conventional CPS
and MLBC showed certain observations in different
diagnostic categories. Both the methods showed same
number of normal smears. Inflammatory smears diagnosis
was more by CPS than by MLBC (42%). However, diagnosis
of Bacterial Vaginosis (14%) and low grade squamous
intraepithelial lesion (36%) were more by MLBC which is a
significant observation. Also the number of infestations
detected by MLBC method was increased koilocytic atypia,
candidiasis and leptothrix. Cytohistological correlation was
done in 22 cases. Increased detection rate was 150% for low
grade intraepithelial lesion. The rate of concordance with
histology was 77% for CPS and LBC, whereas, it was 86%
for MLBC. The rate of increased detection of LSIL through
MLBC was 150%. (Table 4)
Table 4. Comparison of Classification of Cases by Conventional Pap Smear
(CPS) and Manual Liquid Based Cytology (MLBC) with that by Histo-
Pathological Examination (HPE).
Categories CPS MLBC HPE
Normal smear 14 15 1
Inflammatory Smear (NILM) 42 20 8
Bacterial Vaginosis 7 14 0
LSIL 14 36 8
HSIL 1 1 1
Squamous cell carcinoma 2 2 1
Adenocarcinoma 1 1 1
Menopausal 8 6 1
Unsatisfactory 9 1 0
Infestations
HPV (Koilocyticstypia) 2 3 1
Candidiasis 0 1 0
Leptothrix 0 3 0
Total 100 100 22
In addition, to compare the validity of the two methods, we
estimated sensitivity and specificity of the two considering
HPE as the gold standard for the two important diagnoses,
namely, LSIL and inflammation. In the diagnosis of LSIL,
MLBC was more sensitive than CPS (75% vs. 50%) with
similar specificity (100%). In case of inflammation also,
MLBC was found to perform better being more specific (92%
vs. 77%) with same sensitivity (89%).
To work on the utility of MLBC on ancillary studies a
further study was done between 2011 to 2013. Of 60 cases in
our study, 10 cases had no deposit and cell block diagnosis
could not be offered. This inadequate material may be due to
sampling errors. Of the rest 50 cases with adequate material 7
were neoplastic and 43 were non- neoplastic.
43 non-neoplastic lesions include chronic polypoidal
endocervicitis (CPEC - 10 cases), Koilocytes (3 cases),
chronic cervicitis (12 cases), normal study (10 cases), acute
on chronic cervicitis (7 cases), only hemorrhage (1case).
Neoplastic lesions included squamous cell carcinoma SCC
(1 case), dysplasia (2 cases), carcinoma in situ (4 cases)
10 cases of the cell blocks had no deposits. Of these only 3
cases were reported as satisfactory on conventional smears
and 7 cases were unsatisfactory on conventional smears and
had inadequte samples for MLBC smear.This shows that
adequte sampling procedure plays a major role for the better
results by cell blocks. (Table 5)
Table 5. Morphological distribution of cases diagnosed on cell block.
SI.NO Cell block diagnosis No of cases percentage(%)
1 SCC 1 1.7
2 CIN 3 1 1.7
3 CIN 2 2 3.3
4 CIN 1 1 1.7
5 Dysplasia 2 3.3
6 Chronic cervicitis(CC) 12 20
7 CPEC 10 16.7
10 Normal study 10 16.7
8 Acute on chronic
cervicitis(ACC) 7 11.7
9 Koilocytes 3 5
10 Only hemorrhage 1 1.7
11 No deposit 10 16.7
12 Total 60 100
Cases reported as No deposits were maximum when
MLBC had inadequate material and were minimum when
LBC smears were not done and sediment was directly
processed for cell blocks.
3.5. Comparison of Neoplastic Lesions Diagnosed on Cell
Blocks with CPS and MLBC
7 cases of cell blocks were diagnosed as neoplastic
including carcinoma in situ. They included squamous cell
carcinoma SCC (1 case), dysplasia (2 cases), carcinoma in
situ (CIN) (4 cases). SCC was diagnosed as HSIL in CPS and
LBC. 1 case of dysplasia was diagnosed as inflammatory on
CPS and MLBC and other case of dysplasia was diagnosed
as HSIL on CPS and MLBC. 1 case of CIN was
unsatisfactory on CPS and 3 cases of CIN were diagnosed as
HSIL and LSIL.
This shows that cellular architecture is better preserved
and neoplastic lesions which are underdiagnosed on CPS and
MLBC can be correctly diagnosed on cell blocks.
3.6. Comparison of Cell Block Diagnosis with
Histopathology
Histopathologic correlation was obtained in 19 cases, of
these 4 cases were neoplastic and 15 cases were non
neoplastic. Of 19 cases, cell block diagnosis correlated with
histopathology in 14 cases. (Table 6)
Table 6. Histopathological correlation of cell blocks and CPS.
SL NO. Histopathological Correlation Cell blocks CPS
1 Yes 14 (74%) 10 (53%)
2 No 5 (26%) 9 (47%)
3 Total 19 (100%) 19(100%)
Cancer Research Journal 2016; 4(4): 58-68 64
3.7. Statistical Analysis
Sensitivity and specificity of conventional smears, LBC
smears and cell blocks were calculated and compared.
� Positive predictive value for diagnosing neoplastic
lesions on cell blocks: 75%
� Efficacy of cell blocks in the diagnosis of neoplastic
lesions: 89%
� Concordance Rate of CB/Histopathology Vs
CPS/Histopathology is 74% vs 54%.
4. Discussion
Although cervical cancer was historically commoner than
EC, cytological screening has progressively decreased its
prevalence. On the contrary, EC shows an increasing trend. In
developed countries, where cervical cancer screening is
performed, it is estimated that the number of women who die
from EC is almost two fold higher than the number of women
who die from cervical cancer [21]. Unfortunately, although
screening strategies for cervical cancer continue to improve,
screening for EC with cytological techniques remains widely
unaccepted by the medical community.
82 cases with abnormal bleeding history were sampled and
smears obtained in the above mentioned way. These 82 cases
of MLBC were also prepared and compared with the
corresponding conventional pap smears. Cytology smear
results for the 52 patients out of 82 cases showed endometrial
cells. Among 52 cases, histopathological correlation was
obtained in 39 cases.
More recent abstracts reported endometrial cells of 1.8%
and 0.5% on Pap tests in women 40 years and older [22, 23].
Though many of these women with endometrial cells on Pap
tests (61%) had their Pap test collected in the first 14 days of
the menstrual cycle [22].
� However, the various studies published since 1970
shows were listed below- (Table-7)
Table 7. Pap smear studies published since 1970 in women with endometrial
malignancies (35).
Study year Total
no.
Normal
results
Abnormal
results BEC
Burk et al"[24]". 1974 154 123 31 unknown
Vuopala et al"[25]" 1977 88 9 79 abnormal
Bibbo et al"[26]" 1979 33 10 23 normal
Lozowski et al"[27]" 1986 58 23 35 abnormal
Kuebler et al"[28]" 1989 34 12 22 Unknown
Larson et al"[29]" 164 94 94 70 Normal
Zuna and Erroll et al"[30]" 1996 61 25 36 unknown
Eddy et al"[31]" 1997 112 59 53 Abnormal
Fukuda et al"[32]" 1999 99 68 31 Normal
Gu et al."[33]" 2001 76 34 42 unknown
Morimura et al"[34]" 2002 53 15 38 normal
DuBeshter et al."[35]" 2003 300 109 191 abnormal
Brown et al."36]" 2005 412 139 273 Abnormal
Thrall et al."[37]" 2007 73 41 32 abnormal
Present study 2012 52 38 14 abnormal
� The reported prevalence of normal endometrial cells in
all smears varied between 0.1% to 1.4% [38] and
Atypical glandular cells ranging from 0.18 to 0.74%
[39], [40]
� The increased prevalence of endometrial cells in our
study may be because of confinement of patients
selection was only from bleeding history cases in which
some pathology must be there.
The presence of endometrial glandular cells was found to
indicate a five times greater risk of endometrial disease [41].
However, evaluation of prevalence of endometrial
pathology might be complicated because of selection bias
and no adequate control for comparison.
Previous studies showed Endometrial cells on Pap tests
have been associated with normal endometrial findings in 22-
97% of women, polyps in 1-41%, hyperplasias 1-20%,
atypical hyperplasia in 0.6-8%, and carcinoma in 1-15% [42,
43, 44].
In our study, we had maximum of cases with leiomyoma
with proliferative endometrium in 8 (20.5%) cases, followed
by endometrial hyperplasia without atypia in 6 (15.4%)
cases, adenomyomatous polyps in 4 (10.3%) cases and
proliferative endometrium, disordered proliferative
endometrium and endometritis in 3 (7.7%) cases each.
There were 8 (20.5%) cases of malignancy, and 1 (2.6%)
case of precursor lesion with complex hyperplasia with
atypia. So, the final diagnosis was completely benign
pathology in 31 patients and carcinoma in 8 patients.
Among all benign lesions, leiomyoma with proliferative
endometrium was most common because, leiomyoma is
found to be the most common pathology associated with
abnormal uterine bleeding by causing inadequate contraction
of myometrium and irregular spotting leads to shedding of
endometrium.
Recently, Obenson and coworker reported that their most
common benign lesions were endometrial polyps [45]. While
in our study, adenomyomatous polyp was third most common
lesion preceded by simple hyperplasia.
Sensitivity of Pap testing for true glandular neoplasia is
less than that for squamous lesions, in part because glandular
lesions arise in the endocervical canal and endometrial cavity
where they may be difficult to sample.
The low sensitivity for endometrial malignancy was
improved only marginally (from 38.3% to 43.8%) by the
addition of the 2001 Bethesda System reporting category of
benign endometrial cells in women aged >40 years, but
specificity of the Pap test for the diagnosis of endometrial
hyperplasia or malignancy decreases from 99.8% to 96% [31].
Available literature suggested compared with conventional
cytology, LBC may be associated with a higher prevalence of
NECs because of more consistent use of sampling
instruments for LBC with better access to this area [23].
The ThinPrep Pap test has high sensitivity and specificity in
detecting or suggesting the presence of endometrial cancer.
Certain cytomorphologic features are helpful in distinguishing
benign and malignant endometrial lesions [32].
Schorge et al., using ThinPrep, demonstrated a sensitivity
65 Nandini N. Manoli et al.: Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
of 65.2% among 45 patients with endometrial cancer [33].
In our study we also found that the MLBC method was
comparable to the CPS. Though the number of inadequate
specimen for MLBC was 5 whereas 1 for conventional pap
smear, the sensitivity, specificity, negative and positive
predictive value was better for MLBCs. Even on cross
tabulation, MLBC showed better histopathological
correlation than conventional pap smear.
MLBC showed a slightly higher concordance coefficient
(0.572) than CPS (0.556)
The advantages of MLBC are increased detection of
cellular abnormalities. This is because of many factors like
adequacy of sample, type of spatula used to collect the
sample, type of sampling like whether direct to vial or split
sample method. Also the polymer solution which is prepared
in our laboratory contains agarose, polyethylene glycol,
alcohol and poly-L-lysine, form a thin monolayer of cells
within a clean background [15, 18].
This helps to detect nuclear changes in readable clean
background MLBC method which we follow helps to bring
in a cheap method. In addition the residual specimen can be
used for ancillary testing like immunocytochemistry (IHC)
on cell block and detection of HPV DNS by PCR or insitu
hybridization [15, 1].
IHC markers which can be studied are p16inK4a, MIB-1,
BD-ProExC and L1 [48].
To continue with the study of advantages of MLBC we
took up a study on cell block and immunocytochemistry on
cell block between the periods of 2011 to 2013.
Cell blocks can be prepared from all types of cytological
specimens, except preparations with low cellularity such as
cerebrospinal fluids. There are several techniques to produce
cell blocks, such as cytocentrifugation, either with direct
formalin fixation or fixation after addition of plasma-
thromboplastin [49].
In addition, there are commercially available systems, such
as the 'Shandon Cell block Preparations System', which offers
a standardized technique with a high reproducibility. Cell
blocks perform in a highly reproducible way when stained
with most antibodies, except for some used in the work- up
of lymphoid lesions. One distinct advantage of cell blocks is
that many slides can be prepared for extensive panels of
immunostains. In addition, the quality control of cell block
staining is identical to that of histopathology. The
morphology of cell blocks is identical to that seen in
histological specimens and therefore familiar to most
pathologists [50].
5. Uses of Cell Block Are
1. It improves the diagnosis of malignant conditions of
cervix due to better presentation of cytomorphologic
features, better staining characteristics of the nucleus,
nucleoli and cytoplasm, clear recognition of nuclear and
cytoplasmic features [51, 52] (Table 8)
Table 8. Sensitivity and specificity of cell blocks in diagnosing neoplastic
lesions of cervix.
Authors Sensitivity (%) Specificity (%)
Gangane Net al(2007)"[53]" 86.3 100
Gupta.S et al(2007)"[54]" 87.5 100
Catteau X (2012)"[55]" 50 100
Present study 75 93
2. Thus, if used in adjunct of the smear it aids in providing
a reliable diagnosis of cervical cancer in majority of
clinically suspected cases and thus the biopsy need can
be reduced significantly in resource poor settings [54]
• we also found also similar advantages in our study
which were observed by Wei Q et al [56] who like
us also found that cell block increased both
sensitivity and specificity.
3. Cell block can be used to do IHC on suspicious cases
which were done in our study. Stains used were
p16inK4A and KI67. It was found that both markers
increased their expression as the grade of dysplasia
increased and maximum in cervical cancer. Thus
helping in improving diagnostic accuracy of cervical
lesion. These were consistent with findings of Siddham
V. B et al [57] for p16inK4a and by Sahebali s et al [58]
for KI67.(Table 9,) FIG 6
There are many studies done on markers and
endometrium. Ghosh et al have found that there was
increased expression of Cyclin D1 and Ki-67 in patients
with endometrial carcinoma relative to proliferative
endometrium and simple hyperplasia, but there was no
such difference between cases of atypical hyperplasia
and endometrial carcinoma. Cyclin D1 expression had a
positive correlation with Ki-67 expression. Cyclin D1
together with Ki-67 may be a marker for endometrial
carcinogenesis and tumor cell proliferation [59].
Other marker studies on cervix have been undertaken p16
and Survivin expression can be correlated with the clinico-
pathological and prognosis of cervical cancer. p16 protein is
found to be associated with CIN grade and lymph node
metastasis. Survivin protein also is the same. Both together
were positively correlated with cervical cancer (r=0.854,
p,0.001) and associated with poor prognosis of cervical
cancer [60].
Table 9. Table showing the different categories of cervical lesions and
markers.
Categories of lesions Number of
lesions(60) P-16 Ki-67 Ecadherin
Squamous cell carcinoma 2 ++ ++ _
Dysplasia 21 + + _
Inflammatory conditions 20 _ _ +
Normal smears(NILM) 17 _ _ ++
Cancer Research Journal 2016; 4(4): 58-68 66
a b
c d
Fig. 6. a. cell block of normal cervix. (H&E, x200) b. p16 ink4a negative on cell block of normal cervix (x200) c. cell block of cervix-HSIL (H&E, X200) d.
p16ink4a positive on cell block of HSIL (H&E, X200).
6. Conclusion
Women are vulnerable group to many illnesses.
Gynecological pathology with conditions related to
Endometrium and cervix are major causes of the health
problems ranging from infections to neoplasia which require
early detection and treatment.
The commonly used method of conventional Pap smear
(CPS) which was used as an screening method has its
limitations. It has many obscuring factors like blood and
inflammatory cells which limited the sensitivity and
specificity of the test.
Liquid based cytology (LBC), a methodology was
introduced in the advanced countries to overcome these
limitations by making a thin monolayer of cells with a clear
background. This method but is costly for developing
countries where awareness, sources for health care are scarce.
To obviate this, a Manual Liquid based cytology method
(MLBC) was introduced in our setup.
It has been found to be useful in both Endometrial and
cervical cytology in increasing the detection of infection and
neoplastic conditions of female genital tract. It is also a cost
effective method which can be used to test ancillary
techniques of MLBC like cell block study, immunocyto
chemistry and HPV testing.
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